A fiber array spectral translator (“FAST”) system when used in conjunction with a photon detector allows massively parallel acquisition of full-spectral images. A FAST system can provide rapid real-time analysis for quick detection, classification, identification, and visualization of the sample. The FAST technology can acquire a few to thousands of full spectral range, spatially resolved spectra simultaneously. A typical FAST array contains multiple optical fibers that may be arranged in a two-dimensional array on one end and a one dimensional (i.e., linear) array on the other end. The linear array is useful for interfacing with a photon detector, such as a charge-coupled device (“CCD”). The two-dimensional array end of the FAST is typically positioned to receive photons from a sample. The photons from the sample may be, for example, emitted by the sample, reflected off of the sample, refracted by the sample, fluoresce from the sample, or scattered by the sample. The scattered photons may be Raman photons.
In a FAST spectrographic system, photons incident to the two-dimensional end of the FAST may be focused so that a spectroscopic image of the sample is conveyed onto the two-dimensional array of optical fibers. The two-dimensional array of optical fibers may be drawn into a one-dimensional distal array with, for example, serpentine ordering. The one-dimensional fiber stack may be operatively coupled to an imaging spectrograph of a photon detector, such as a charge-coupled device so as to apply the photons received at the two-dimensional end of the FAST to the detector rows of the photon detector.
One advantage of this type of apparatus over other spectroscopic apparatus is speed of analysis. A complete spectroscopic imaging data set can be acquired in the amount of time it takes to generate a single spectrum from a given material. Additionally, the FAST can be implemented with multiple detectors. The FAST system allows for massively parallel acquisition of full-spectral images. A FAST fiber bundle may feed optical information from its two-dimensional non-linear imaging end (which can be in any non-linear configuration, e.g., circular, square, rectangular, etc.) to its one-dimensional linear distal end input into the photon detector.
Given the advantageous ability of a FAST system to acquire hundreds to thousands of full spectral range, spatially-resolved spectra, such as Raman spectra, substantially simultaneously, a FAST system may be used in a variety of situations to help resolve difficult spectrographic problems such as the presence of polymorphs of a compound, sometimes referred to as spectral unmixing.
Chemical images may generally be acquired using one of two classes of approaches: (1) scanning, and (2) widefield chemical imaging. In scanning methods, a radiation source is focused onto the surface of a sample and a spectrum from each spatial position is collected using a dispersive spectrograph or interferometer. Long data collection times are common with scanning methods since the duration of the experiment is proportional to the number of image pixels. Because of such long data collection times, scanned images are captured at low image definition, which relates directly to the limited utility of the technique as an imaging tool for the routine assessment of material morphology. Furthermore, the spatial resolution of the image is limited by the size of the source illumination on the sample and the rastering mechanism, which requires the use of moving mechanical parts that are challenging to operate reproducibly. In addition, for light-absorbing materials, scanning methods present an enormous challenge. These materials have low damage thresholds, requiring the use of low laser power densities to minimize local thermal expansion and sample degradation.
Despite the limitations, scanning methods are relatively mature techniques and have been applied in a number of applications. An advantage of scanning-based chemical imaging is the ability to capture the entire spectrum in an efficient manner. This advantage is best realized in the research evaluation of new material systems where the underlying spectroscopy is not well understood, and therefore, benefits may be available from the analysis of the entire spectrum.
In widefield chemical imaging, the entire sample field of view is illuminated and analyzed simultaneously. Numerous widefield chemical imaging approaches have been demonstrated, with the majority of methods involving the recording of an image at discrete spectral intervals though an imaging spectrometer (i.e., LCTF (Liquid Crystal Tunable Filter), AOTF (Acousto-Optic Tunable Filter), etc.).
Because both (X-Y) spatial dimensions are collected simultaneously in widefield Chemical Imaging using imaging spectrometers, the experiment duration is proportional to the number of spectral channels and not to the number of image pixels. The widefield advantages are best realized when high fidelity images at a limited number of wavelengths provide sufficient chemical and spatial information. In most materials characterization applications, only a limited number of spectral bands (typically<100) are required to analyze the analytes of interest. By reducing the number of spectral channels, the duration of the widefield experiment decreases without losing spatial resolution. In addition, time-dependent changes in the sample are only observed in the spectral dimension, which simplifies the flatfielding or analysis of chemical images in widefield imaging.
Conversely, attempts to reduce the duration of scanning experiments (in the scanning approach discussed above) compromise either the spatial resolution or the field of view. Reducing the number of spectral channels in scanning mode has little effect on the experiment duration since the entire chemical spectrum is captured simultaneously (in the scanning approach discussed above). Scanning experiments record time dependent sample changes as spatial variations. Pixels collected at different times often have induced spectral differences that complicate flatfield correction.
A phenomenon of Raman spectroscopy of crystalline materials (e.g., polymorphs) is the effect the crystal orientation (with respect to incident and scattered light) has on the resultant spectrum. The crystal orientation-dependent effects on the Raman spectra manifest themselves as changes in the relative band intensities and/or frequency positions. For a plurality of crystals that has a random orientation, the Raman spectrum of a single crystal can potentially be much different than a spectrum of the bulk material. This phenomenon can result in a false conclusion that the single crystal is a different polymorph than the reference material. This effect can be lessened by reducing the degree of polarization of the excitation illumination as well as minimizing the polarization dependence of the spectrometer.
Current Raman well-plate polymorph screening instruments typically involve the acquisition of Raman data in a semi-automated or fully-automated fashion. These instruments are typically configured in a point scanning format in which a laser beam is focused in a small spot in an attempt to localize the illumination and collection from a single crystal. Semi-automated scanning Raman analysis is typically first preceded with an optical (i.e., brightfield and/or polarized light microscopy) means of viewing the wells in the well-plate. A user then manually selects regions of interest followed by a subsequent automated Raman dispersive acquisition of those selected regions. This approach is susceptible to human subjectivity in targeting appropriate crystals for subsequent analysis. On the other hand, in a fully-automated configuration, a single or multipoint acquisition is performed in a blind fashion within each well of the well-plate. The acquisition time of the experiment in each case is proportional to the number of measurements acquired per well.
For most spectral unmixing methods to be effective, a minimum of 6-12 spectra must be acquired having some spectral variability representative of the compositional variance within the sample. To support this quantity of measurements using traditional Raman screening methods would result in extremely long experimental acquisition times since data is normally collected in a serial fashion.
The present disclosure provides methods and systems for overcoming the above-mentioned limitations of the prior art. In certain embodiments, the present disclosure describes a system and a methodology that each combine Raman spectroscopy performed in a manner that utilizes widefield illumination, simultaneous multipoint Raman spectral acquisition, and spectral unmixing for the purpose, for example, of high throughput polymorph screening. The use of FAST enables full spectral acquisition for hundreds to thousands of spatially resolved spectra in a single image frame—dramatically increasing data acquisition rates compared to current tunable filter based technologies. Software, hardware, and/or a combination of software and hardware may be used to extract the spatial/spectral information to reconstruct hyperspectral (chemical imaging) data cubes of the original object and/or determine the presence and/or quantities (actual or relative) of polymorphs present in a sample. Furthermore, FAST is a rugged technology that operates over an extensive spectral range from ultraviolet (UV) to infrared (IR).
Accordingly, it is on object of the present disclosure to provide a method for polymorph screening, comprising illuminating a sample using widefield illumination to thereby produce scattered photons, which may be Raman scattered photons; receiving the scattered photons substantially simultaneously from a plurality of spatial locations of the sample using a fiber array spectral translator and directing the scattered photons to a photon detector, where each fiber of the fiber array spectral translator may receive photons from a different region of the sample; detecting the scattered photons and providing therefrom plural spectra of the sample, which may be Raman spectra; and applying a spectral unmixing algorithm to the plural spectra to thereby determine the presence of one or more polymorphs in the sample.
It is another object of the present disclosure to provide a system for polymorph screening, comprising a photon source for illuminating a sample using widefield illumination to thereby produce scattered photons, which may be Raman scattered photons; a fiber array spectral translator for receiving the scattered photons substantially simultaneously from a plurality of spatial locations of the sample and directing the scattered photons to a photon detector, where each fiber of the fiber array spectral translator may receive photons from a different region of the sample; the photon detector for detecting the scattered photons and providing therefrom plural spectra of the sample, which may be Raman spectra; and a-microprocessor unit for applying a spectral unmixing algorithm to the plural spectra to thereby determine the presence of one or more polymorphs in the sample.
It is a further object of the present disclosure to provide a method for polymorph screening, comprising: illuminating a mixture with first photons in a widefield illumination manner to thereby produce second photons, such as Raman scattered photons, wherein the sample comprises a polymorph of a compound wherein first ones of the polymorph are disposed in a first orientation and second ones of the polymorph are disposed in a second orientation, and wherein first ones of the second photons are scattered from the first oriented polymorphs and second ones of the second photons are scattered from the second oriented polymorphs; receiving the second photons at a proximal end of a fiber array spectral translator comprising plural fibers wherein each fiber of the fiber array spectral translator is associated with a different predetermined region of the sample, where the regions may overlap; delivering the second photons at a distal end of the fiber array spectral translator to a photon detector; detecting the second photons and providing therefrom plural spectra, such as Raman spectra, comprising a first spectrum of the first oriented polymorphs and a second spectrum of the second oriented polymorphs; and applying a spectral unmixing algorithm to the plural spectra to thereby determine a quantity of the first and second oriented polymorphs.
It is yet a further object of the present disclosure to provide a system for polymorph screening, comprising a photon source for illuminating a mixture with first photons in a widefield illumination manner to thereby produce second photons, such as Raman scattered photons, wherein the sample comprises a polymorph of a compound wherein first ones of the polymorph are disposed in a first orientation and second ones of the polymorph are disposed in a second orientation, and wherein first ones of the second photons are scattered from the first oriented polymorphs and second ones of the second photons are scattered from the second oriented polymorphs; a fiber array spectral translator comprising plural fibers for receiving the second photons at a proximal end wherein each fiber of the fiber array spectral translator is associated with a different predetermined region of the sample, and for delivering the second photons at a distal end to a photon detector; the photon detector for detecting the second photons and providing therefrom plural spectra, such as Raman spectra, comprising a first spectrum of the first oriented polymorphs and a second spectrum of the second oriented polymorphs; and a microprocessor unit for applying a spectral unmixing algorithm to the plural spectra to thereby determine a quantity of the first and second oriented polymorphs.
It is still a further object of the present disclosure to provide a method for polymorph screening, comprising illuminating a sample with first photons in a widefield illumination manner to thereby produce second photons, such as Raman scattered photons, wherein the sample comprises a plurality of polymorphs of a compound wherein first ones of the second photons are scattered from a first polymorph and second ones of the second photons are scattered from a second polymorph; receiving the second photons at a proximal end of a fiber array spectral translator comprising plural fibers wherein each fiber of the fiber array spectral translator is associated with a different predetermined region of the sample; delivering the second photons at a distal end of the fiber array spectral translator to a photon detector; detecting the second photons and providing therefrom plural spectra, such as Raman spectra, comprising a first spectrum of the first polymorph and a second spectrum of the second polymorph; and applying a spectral unmixing algorithm to the plural spectra to thereby determine a quantity of each of the first and second polymorphs. Furthermore, first ones of the first polymorph may be disposed in a first orientation and second ones of the first polymorph may be disposed in a second orientation wherein a first subset of the first ones of the second photons are scattered from the first oriented polymorphs and a second subset of the first ones of the second photons are scattered from the second oriented polymorphs. Additionally, the first spectrum may comprise a third spectrum from the first oriented polymorphs and a fourth spectrum from the second oriented first polymorphs. Moreover, the spectral unmixing algorithm may also determine a quantity of the first oriented polymorphs and a quantity of the second oriented polymorphs.
It is another object of the present disclosure to provide a system for polymorph screening, comprising a photon source for illuminating a sample with first photons in a widefield illumination manner to thereby produce second photons, such as Raman scattered photons, wherein the sample comprises a plurality of polymorphs of a compound wherein first ones of the second photons are scattered from a first polymorph and second ones of the second photons are scattered from a second polymorph; a fiber array spectral translator comprising plural fibers for receiving the second photons at a proximal end wherein each fiber of the fiber array spectral translator is associated with a different predetermined region of the sample, and for delivering the second photons at a distal end to a photon detector; the photon detector for detecting the second photons and providing therefrom plural spectra, such as Raman spectra, comprising a first spectrum of the first polymorph and a second spectrum of the second polymorph; and a microprocessor unit for applying a spectral unmixing algorithm to the plural spectra to thereby determine a quantity of each of the first and second polymorphs. Furthermore, first ones of the first polymorph may be disposed in a first orientation and second ones of the first polymorph may be disposed in a second orientation wherein a first subset of the first ones of the second photons are scattered from the first oriented polymorphs and a second subset of the first ones of the second photons are scattered from the second oriented polymorphs. Additionally, the first spectrum may comprise a third spectrum from the first oriented polymorphs and a fourth spectrum from the second oriented polymorphs. Moreover, the microprocessor unit may apply the spectral unmixing algorithm to determine a quantity of the first oriented polymorphs and a quantity of the second oriented polymorphs.